Mutations in the gene for Leucine-Rich Repeat Kinase 2 (LRRK2) are responsible for an autosomal dominant form of Parkinson's Disease (PD). Biochemical analyses of LRRK2 in cell lysates indicate that the protein dimerizes, and that impairment of kinase activity results in depletion of dimers and formation of higher-order oligomers. Interestingly, PD-associated mutations shift the balance toward the dimeric (active) state of LRRK2. Because LRRK2 kinase activity is apparently dependent on its oligomerization state, we propose in Aim 1 to elucidate the process of LRRK2 self-association in living cells using Fluorescence Fluctuation Spectroscopy (FFS) approaches. Three modes of FFS will be employed: 1. The Number and Brightness (N&B) mode will reveal the oligomeric states and estimate the affinities of LRRK2 and LRRK2 mutants throughout the cell; 2. The two-color cross-correlation mode will be used to detect hetero-oligomerization between wild-type and mutant forms of LRRK2 as a means of validating this approach for investigations of hetero-interactions between LRRK2 and its binding partners; 3. FFS in the Total Internal Reflection Fluorescence (TIRF) mode will allow us to focus specifically on LRRK2 oligomerization on the plasma membrane. These methods will be used to determine how LRRK2 self-association is affected by interventions that affect its phosphorylation state, its kinase and GTPase activities, and its state of palmitoylation, a modification of LRRK2 recently identified in our laboratory. Density gradient centrifugation, kinase and GTPase assays, and cell toxicity analyses will be performed in parallel to characterize the effects of these interventions on the properties of LRRK2 and LRRK2- expressing cells. Aim 2 will be directed at elucidating potential interactions between LRRK2 and a-synuclein, the major component of the pathognomonic neuronal inclusions of PD (Lewy bodies). We recently observed that LRRK2 increases the level of a-synuclein phosphorylated at serine 129, which is a selective marker of pathology in human PD brains. Most strikingly, we found that phosphorylated a-synuclein significantly increases LRRK2 abundance and toxicity in transfected cells. Therefore, we propose to use FFS approaches to define the nature of complexes formed between LRRK2 and a-synuclein, either in the cytosol or on membranes. Taken together, our studies will define the biophysical states of LRRK2 that are associated with cell toxicity and with high and low levels of kinase and GTPase activity, and will elucidate the interplay between pathogenic mechanisms of LRRK2 and a-synuclein mutations.